The invention relates to a method for producing cathine.
Cathine ((1S,2S)-norpseudoephedrine) can be extracted from the leaves of khat and is frequently used as an appetite suppressant. Due to the sympathomimetic function stimulating the autonomic nervous system, it is used as a cardiovascular drug.
A variety of methods are already known from the prior art for synthetic production.
A 7-step synthesis is known, for example, from the publication “Stereoselective Synthesis of Norephedrine and Norpseudoephedrine by Using Asymmetric Transfer Hydrogenation Accompanied by Dynamic Kinetic Resolution” by Hyeon-Kyu Lee at al. in the Journal of Organic Chemistry 2012, pgs. 5454-5460.
The publication “Efficient Synthesis of Ephedra Alkaloid Analogous Using an Enantiomerically Pure N-[(R)-(+)-α-Methylbenzyl]aziridine-2-carboxaldehyde” by Gwon-II Hwang at al. from J. Org. Chem, 1996, 61, 6183-6188, discloses a method for producing cathine with high enantiomeric excess, which is based on an enantiomerically pure substrate and is very expensive.
Other methods, as described, for example, in the publication “Asymmetric N1 Unit Transfer to Olefins with a Chiral Nitridomanganese Complex: Novel Stereoselective Pathways to Aziridines or Oxazolines” by Masaaki Nishimura at al., disclose methods in which cathine is formed as a by-product.
It is therefore the object of the invention to make a simple method for producing cathine available, which overcomes the disadvantages of the state of the art, requires only few reaction steps, is inexpensive, results in high enantiomeric and diastereomeric purity and in a high yield, and requires as few processing steps as possible. The method should be easily scalable.
Using the method according to the invention, cathine can be produced in enantiomeric purity of >99% and diastereomeric purity of 70%, and in one advantageous embodiment of up to 97%. Conversion of 90% can be achieved. For this purpose, commercially available starting materials may be used. The method necessitates few processing steps, is easy to scale, and can be carried out as a one-pot reaction.
The invention will be described hereinafter in the general form thereof.
(S)-selective enzyme within the meaning of the invention is an enzyme that results in an (S)- configured product.
In a first reaction step, benzaldehyde is reacted in vitro with an acetyl donor according to formula (1)
by way of an (S)-selective lyase to yield an enantiomer mixture of the compounds according to formulas (2) and (3)
wherein the abbreviation PAC denotes phenylacetylcarbinol.
The acetyl donor according to formula (1) can be acetaldehyde where R═H or pyruvate where R═COOH.
The reaction pathway is shown by way of example in FIG. 1.
The lyase may be, for example, an (S)-selective lyase from Acetobacter, in particular Acetobacter pasteurianus, in particular the ApPDC-E4690 variant.
Preferably, purified and lyophilized enzymes are used. Lyophilization has the advantage that the enzymes are stable and high enzyme concentrations can be used; in addition, purified enzymes result in considerably higher optical purities, which is to say high enantiomeric and diastereomeric excess of the product.
Furthermore, cofactors can be used for the (S)-selective lyase, which increase the conversion.
For example, magnesium ions such as magnesium sulfate or magnesium chloride, together with thiamine diphosphate can be used as cofactors.
When magnesium sulfate and thiamine diphosphate are used as cofactors, a concentration range of 1 to 5 mM, and preferably 2.5 mM, for magnesium sulfate and of 5 to 300 μM, and preferably 100 μm, for thiamine diphosphate is preferred.
The reaction can be carried out at a pH value of 5 to 8, and preferably of 6 to 7.5.
For this purpose, a potassium phosphate buffer can be used, for example. However, buffers such as HEPES, MOPS, TEA or TRIS-HCl are also possible.
The preferred temperature range is room temperature, but the reaction can also be carried out well between 20° C. and 30° C., and more particularly 25° C.
The reaction is preferably carried out at atmospheric pressure.
As an alternative, the enzymatic reaction can take place in vivo. This has the advantage that the enzyme can be produced cost-effectively as a catalyst.
For this purpose, E. coli. bacteria can be used as production organisms.
Genes coding for a lyase in the first step can be ligated into a vector.
The E. coli strains are preferably recombinant and contain plasmids that carry genes for an (S)-selective lyase.
The plasmids can preferably include genes for the above-mentioned lyases.
For example, pET22b or pKK233 base structures, which include the corresponding lyase genes, can be used as plasmids.
The production organisms secrete the desired product according to formulas (2) and (3) into the aqueous solution.
In a preferred embodiment of the method according to the invention, the undesirable compound according to formula (2)
is reacted by way of a benzaldehyde lyase to yield benzaldehyde and acetaldehyde.
The reaction scheme is shown in FIG. 2.
This has the advantage that the undesirable by-product according to formula (2) is cleaved, and the resultant benzaldehyde and acetaldehyde are returned to the process, increasing the yield of the desired product according to formula (3). The enantiomeric excess of (S)-PAC according to formula (3) can be increased in total to >97%. The yield in this step is 95%, based on the total amount of PAC used.
In the reaction of the compound according to formula (2), it is advantageous to add an excess of the acetyl donor during the reaction process, so that sufficient acetyl donor is available for the further reaction to yield the desired intermediate compound according to formula (3). This is advantageous in particular with volatile acetyl donors, such as acetaldehyde. For example, a 10-fold excess of acetyl donor compared to the benzaldehyde can be used.
The intermediate product is preferably separated off. For this purpose, column chromatography with silica gel can be used, using petroleum ether:ethyl acetate (90:10) as the separating liquid.
The compound according to formula (3) thus obtained is reactd in a second reaction step with an amine donor by way of an (S)-selective transaminase to yield the cathine end product ((1S,2S)-norpseudoephedrine). The reaction is shown schematically in FIG. 3.
Transaminases which convert the substrate and are (S)-selective can be used for the second reaction step, which is the chemical reductive amination. The transaminase can be from Chromobacterium, such as Chromobacterium violaceum, preferably CV2025. Furthermore, transaminases from Alcaligens denitrificans, Arthrobacter citreus, Bacillus megaterium, Pseudomonas fluorescens, Vibrio fluvialis or Caulobacter crescentus may be used.
Preferably, purified and lyophilized enzymes are used. Lyophilization has the advantage that the enzymes are stable and high enzyme concentrations can be used; in addition, purified enzymes result in considerably higher optical purifies, which is to say high enantiomeric and diastereomeric excess of the product.
(S)-alpha-methylbenzylamine, benzylamine, isopropylamine, L-alanine, (±)-1-methyl-3-phenylpropylamine, (±)-1-aminoindane can be used as amine donors, for example.
Pyridoxal 5′-phosphate can be used as a cofactor.
The pyridoxal 5′-phosphate concentration preferably ranges between 100 and 200 μM.
The reaction can be carried out in an aqueous medium in a pH range of 6 to 11, and preferably of 7.5 to 8.5.
Suitable buffers, such as HEPES, potassium phosphate, MOPS, TEA or TRIS-HCl, can be used for this purpose.
The preferred temperature range is 25° C., but the reaction can also be carried out well in a range of 20° C. to 30° C.
The reaction is preferably carried out at atmospheric temperature.
As an alternative, the enzymatic conversion with the (S)-selective transaminase can take place in vivo.
For this purpose, E. coli bacteria can be used as production organisms.
Genes coding for a transaminase can be ligated into a vector.
The E. coli strains are preferably recombinant and contain plasmids that carry genes for a (S)-selective transaminase.
The plasmids can preferably include genes for the above-mentioned transaminases.
For example, pET29a or pKK233 base bodies, which include the corresponding transaminase genes, can be used as plasmids.
The production organisms secrete the desired product according to formula (3) into the aqueous solution.
The reactions in vivo can be carried out in the method according to the invention either only for the first or the second reaction step, or for both reaction steps 1 and 2. The remaining conditions can be selected for each step in the same manner as for the enzymatic reaction in vitro.
FIG. 4 shows a summary of the steps from FIGS. 1 and 3.